Related enzymes that have a common function may or may not have significant sequence identity. For example, Bacillus alpha-amylases (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) are classified as “Termamyl-like” if their amino acid sequences share 60% or higher identity to B. licheniformis alpha-amylase. See WO 96/23874. Hybrid amylases can be created between amylases sharing 62% or higher sequence identity. See Gray et al., J. Bacteriology 166: 635-43 (1986); U.S. Pat. No. 6,143,708. For example, a chimeric amylase containing residues 1-300 of Bacillus amyloliquefaciens and residues 301-483 of B. licheniformis has been recombinantly expressed and crystallized. See Brzozowski et al., Biochemistry 39: 9099-107 (2000); see also WO 96/23874; WO 97/41213. However, even amylases sharing less than 25% sequence identity, such as the amylases from Bacillus subtilis and B. licheniformis, nevertheless may share a common catalytic function and an overall conserved three-dimensional (3D) fold.
Crystal structures of numerous amylases are currently available. See Brzozowski et al. (2000) supra. The Protein Data Bank (PDB), for example, contains 3D structures of at least the amylases shown in TABLE 1.
TABLE 1PDB Acc. No.Amylase1rp8Barley alpha-amylase1pifPig alpha-amylase1aqmAlteromonas haloplanctis alpha-amylase1hvxBacillus stearothermophilus alpha-amylase1ua7Bacillus subtilis alpha-amylase1mxdPyrococcus woesei alpha-amylase1jaeTenebrio molitor larval alpha-amylase1wzaHalothermothrix orenii alpha-amylase1ud2Bacillus sp. KSM-K38 alpha-amylase2guyAspergillus niger alpha-amylase1smdHuman salivary alpha-amylase1kbbHuman pancreatic alpha-amylase (same as above)1bliBacillus licheniformis alpha-amylase2gjrBacillus halmapalus alpha-amylase1uh2Thermoactinomyces vulgaris R-47 alpha-amylase
Comparison of these crystal structures reveals a high degree of conservation of three-dimensional (3D) structure, even in the absence of significant sequence similarity. All reported alpha-amylase structures share a (β/α)8 catalytic core domain, domain A. “(β/α)8” refers to a so-called “TIM barrel structure,” defined as a conserved protein 3D conformation, or “fold,” consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone. See Lolis et al., Biochemistry 29: 6609-18 (1990). The B domain is an excursion, or extended structure, between the barrel strand β-3 and helix α-3 of domain A. The C domain, typically an eight-stranded β-sheet, forms the remaining C-terminal portion of the amylases. The domain structure of amylases is described in more detail below.
There is a continuing need in the art to provide variant alpha-amylases that fold properly, maintain stability, and demonstrate efficient expression in a recombinant host cell, without the necessity of making wholesale changes to the amino acid sequence or 3D structure of the protein.